The Effect of Donor Cell Serum Starvation and Oocyte Activation Compounds on the Development of Somatic Cell Cloned Embryos

Abstract

Two experiments, one comparing nuclear transfer (NT) embryo activation compounds, the other donor cell treatments, were conducted with a goal of identifying factors that improve the in vitro development of cloned bovine embryos. In experiment 1, 539 NT embryos were produced by combining serum starved bovine fetal fibroblasts with enucleated in vitro matured oocytes, activated with ionomycin, then randomly allocated to be incubated for 4 hours in either Butyrolactone-I (BL-I) or 6-dimethylaminopurine (DMAP). There was no significant difference in development to blastocyst or compact morula of fused embryos at Day 6.5 between BL-I and DMAP activated embryos (22.4% vs. 20.2%; p = 0.18). Karyotyping of 20 blastocysts and compact morula from each group determined that 65% of BL1 and 63% of DMAP embryos were diploid with the remainder mixoploid (2n + 4n). In Experiment 2, the development of 389 NT embryos reconstructed from either serum starved or serum fed fetal fibroblasts was assessed. More Day 7 blastocysts and compact morula developed in the serum starved group (34.5% vs. 18.8%; p = 0.008). To verify the viability of BL-I activated embryos, 10 blastocytes from experiment 2 were transferred into 4 recipient cows. Two morphologically normal fetuses, genetically identical to the original fetal cell line, were surgically recovered at day 45 of gestation. In summary, serum starvation of bovine fetal fibroblasts prior to NT significantly improved development to blastocyst. Additionally, we have shown that BL-I is a novel alternative compound for use in combination with ionomycin to activate NT embryos.

Clonal Lines of Transgenic Fibroblast Cells Derived from the Same Fetus Result in Different Development When Used for Nuclear Transfer in Pigs1

“…Whereas some authors claim that the use of cells in G0 is required for complete reprogramming [3,4], others used cycling donor cells in presumptive G1 and obtained offspring [12]. In contrast to comparative studies in cattle [7,28], in which the use of serum-starved donor cells resulted in better development, we found no effect of serum starvation on the in vitro development of porcine NT embryos. Therefore, the use of cells in a stage of the cell cycle other than the desired one cannot be excluded.…”

Assessment of Chromosomal Abnormalities in Bovine Nuclear Transfer Embryos and in Their Donor Cells

“…Despite the high rates of embryo and fetal mortalities associated with cloning from somatic cells, few studies of the chromosomes of NT embryos has been reported so far (Hill et al, 2000;Talbot et al, 2000). While Hill et al reported a high rate of polyploid mosaicism, no anomalies were identified in the study performed by Talbot et al The focus of the present study was to assess chromosomal aberrations in bovine NT embryos produced from two different primary somatic cell cultures.…”

Production of Nuclear Transfer-Derived Piglets Using Porcine Fetal Fibroblasts Transfected with the Enhanced Green Fluorescent Protein1

“…The cell-cycle stage is a subject of debate in the production of cloned animals by SCNT because no system thus far provides 100% synchronization of cells in a certain stage [31] and therefore the use of cells in the same cellcycle stages in SCNT procedures cannot be guaranteed. Whereas some researchers assert that the use of cells in G0 is required for complete reprogramming [32][33][34], others used cycling donor cells in presumptive G1 and obtained offspring [14]. In this study, we investigated whether serum starvation, which is a commonly used method to synchronize cell cycle, could improve the SCNT embryo development.…”